Presynaptic protein cast

ABSTRACT

The present invention enabled the detection and quantification of CAST, which is localized to synapses and tightly bound to the cytomatrix, and of the mRNA encoding the CAST. Furthermore, it was revealed that CAST functions as a protein scaffold for localizing RIM1 to synapses, contributing as a molecular basis for active zone formation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-Part of PCT/JP03/02718 filed onMar. 7, 2003, which claims priority from Japanese Application No.2002-063186 filed on Mar. 8, 2002.

FIELD OF THE INVENTION

The present invention relates to a novel protein related to the synapticcytomatrix and which is useful in the medical field.

BACKGROUND OF THE INVENTION

A great number of neurotransmitter receptors and scaffolding moleculessuch as PSD-95 are present in the postsynaptic density (PSD) of neuralsynapses. These molecules play important roles in the formation andstructural maintenance of the PSD. Furthermore, cytomatrix proteins suchas Bassoon and RIM are present in the cytomatrix that constitutes thepresynaptic active zone (i.e., the cytomatrix at the active zone; CAZ).These proteins play crucial roles in the formation and structuralmaintenance of the active zone. The efficient transmission of signalsbetween synapses is extremely important in learning, and in theconstruction and maintenance of memories. Hence, it is easy to envisionthat failure of this process will cause various neuropathic andneurodegenerative disorders.

Numerous molecular groups associated with the molecular mechanism foraxonal guidance of neurons (pathfinding) have been identified byanalyses using flies and nematodes, enhancing the understanding of themechanism to a certain extent. However, in subsequent synaptogenesis,much remains to be clarified as to what kind of molecules are temporallyand spatially involved in the formation of mature synaptic contacts.

SUMMARY OF THE INVENTION

An objective of this invention is to find a novel protein linked to thecytomatrix and considered to play an important role in synaptogenesis.

In order to identify proteins concentrated at the synaptic junction, thepresent inventors prepared crude membrane (P2) fractions and PSDfractions from rat cerebrum, and dissolved these in a buffer containingurea. The dissolved samples were then fractionated using an anionexchange column. Each eluted fraction was subjected to electrophoresisand stained, and the band patterns of P2 and PSD fractions werecompared. Proteins concentrated in the PSD fraction were identifiedusing these band patterns, and mass spectrometry revealed two proteins(p500 and 120 proteins) that were not found in databases. Hereinafter,the phrase “120 protein” refers to the protein of this invention and isalso described as “CAST”.

Amino acid analysis of these proteins revealed that four peptidesequences derived from CAST coincided with the internal sequence of theKIAA0378 protein. The results of further studies showed that the p120protein is detected mainly in the brain and is tightly bound to thecytomatrix. Furthermore, CAST was also revealed to function as a proteinscaffold, localizing to synapses the Rab3A-interacting molecule (RIM1)that plays a critical role in synaptic plasticity (Castillo P. E. etal., Nature 415:327-330, 2002; Schoch S. et al., Nature 415:321-326,2002).

Specifically, the present invention relates to:

[1] a protein selected from the group consisting of:

-   -   (a) a protein having the amino acid sequence set forth in SEQ ID        NO: 2; and    -   (b) a protein localized to synapses and having the amino acid        sequence set forth in SEQ ID NO: 2, wherein one or more amino        acid residues are deleted, substituted, or added;

[2] a DNA encoding the protein of [1];

[3] the DNA of [2] having the nucleotide sequence set forth in SEQ IDNO: 1;

[4] a DNA selected from the group consisting of:

-   -   (a) a DNA having the nucleotide sequence set forth in SEQ ID NO:        1; and    -   (b) a DNA that hybridizes under stringent conditions to a DNA        that has a nucleotide sequence complementary to the nucleotide        sequence set forth in SEQ ID NO: 1, and which encodes a protein        localized to synapses;

[5] a DNA selected from the group consisting of:

-   -   (a) a DNA having the nucleotide sequence set forth in SEQ ID NO:        1; and    -   (b) a DNA having a nucleotide sequence with 90% or more homology        to the nucleotide sequence set forth in SEQ ID NO: 1 and        encoding a protein localized to synapses;

[6] a protein encoded by the DNA of [4] or [5];

[7] a recombinant vector comprising the DNA of any one of [2] to [5];

[8] a transformant resulting from transformation of a host with the DNAof any one of [2] to [5];

[9] a method for producing a protein localized to synapses, wherein saidmethod comprises the steps of:

-   -   (1) culturing the transformant of [8]; and    -   (2) collecting the protein from the culture, wherein the protein        is localized to synapses and expressed by the transformant;

[10] an antibody that reacts with the protein of [1] or [6];

[11] a method for determining the level of the protein of [1] or [6],wherein said method uses the antibody of [10];

[12] a method for measuring the level of a DNA or RNA encoding theprotein of [1] or [6], wherein said method uses as a primer or probe anoligonucleotide comprising at least 15 continuous nucleotides of thenucleotide sequence set forth in SEQ ID NO: 1;

[13] a method for detecting the protein of [1] or [6] using the antibodyof [10];

[14] a method of screening for a substance that reacts with the proteinof [1] or [6], wherein said method comprises the steps of:

-   -   (1) mixing a test substance with the protein of [1] or [6], and        produced by the method of [9] or the transformant of [8]; and    -   (2) measuring the level of the bound or unbound test substance;

[15] a method of screening for a substance affecting the expression ofthe protein of [1] or [6], wherein said method comprises the steps of:

-   -   (1) adding a test substance to cells expressing the protein of        [1] or [6] followed by culturing; and    -   (2) measuring the level of the protein of [1] or [6] which is        expressed in the cells, or mRNA encoding said protein;

[16] a method of screening for a substance affecting the expression ofthe protein of [1] or [6], wherein said method comprises the steps of:

-   -   (1) identifying the promoter region controlling the expression        of the protein of [1] or [6]; and    -   (2) measuring the effect of a test substance on the promoter        activity;

[17] a method of screening for a substance affecting the distribution ofthe protein of [1] or [6], wherein said method comprises the steps of:

-   -   (1) adding a test substance to cells expressing the protein of        [1] or [6] followed by culturing the cells; and        -   (2) determining the distribution of the protein of [1] or            [6];

[18] the protein of [1] that binds to RIM1 and,

[19] an inhibitor for binding of RIM 1 to the protein of [1] comprisinga peptide selected from the group consisting of:

-   -   (1) a peptide comprising the amino acid sequence set forth in        SEQ ID NO: 6; and    -   (2) a peptide comprising the amino acid sequence set forth in        SEQ ID NO: 6 wherein one or several amino acids are deleted,        substituted, or added.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing the electrophoresis band patterns of P2and PSD fractions eluted using a MonoQ anion exchange chromatographycolumn. CAST was specifically recognized in the PSD fraction.

FIG. 2A represents the coiled-coil domains in the amino acid sequence ofCAST; while FIG. 2B is an electrophoretogram showing CAST bands ofendogenous CAST, and CAST expressed from CAST cDNA in rabbitreticulocyte extracts. CAST expressed from cDNA showed the same mobilityas that of the endogenous CAST.

FIG. 3 is a set of photographs showing (A) the organ distribution ofCAST; (B) the distribution of CAST in each cellular fraction; and (C)the solubilization of CAST by non-ionic and ionic surfactants. CAST wasthought to be distributed in synapses and tightly linked to thecytomatrix.

FIG. 4 is a set of photographs showing (A) immunohistostainings of themouse hippocampal region with anti-CAST antibody and anti-synaptotagminI antibody; (B) immunohistostainings of a primary culture of rathippocampal neurons with the anti-CAST antibody, anti-Bassoon antibody,or anti-PS-95 antibody; and (C) immuno-electron micrographs of synapsesstained with the anti-CAST antibody.

FIG. 5 is a series of photographs showing (A) CAST, Bassoon, and RIM1,immunoprecipitated by the anti-CAST antibody; and (B) CAST, Bassoon, andRIM1, immunoprecipitated by the anti-Bassoon antibody. CASTco-immunoprecipitated with Bassoon and RIM1, and it was thought thatthey have a mutual binding activity.

FIG. 6A is a diagram and photograph showing RIM1 co-immunoprecipitatedwith each domain of CAST; and FIG. 6B is a photograph showing CASTco-immunoprecipitated with each domain of RIM1. The C-terminal domain ofCAST was thought to bind to the PSD-95/Discs-Large/ZO-1 (PDZ) domain ofRIM 1.

FIG. 7 is a diagram showing the presence or absence of binding activitybetween whole CAST and RIM1.

FIG. 8 is a series of photographs showing the distribution of the fusionprotein of ECFP and CAST that does not have binding activity withrespect to RIM1 (CAST-2). CAST distribution in the synaptic active zonewas revealed to be independent of the binding activity with RIM 1.

FIG. 9 is a series of photographs showing the distribution of fusionproteins of Myc with whole RIM1 (RIM1), RIM1 deprived of the PDZ domain(RIM1 ΔN), and RIM1's PDZ domain (RIM1 PDZ). The PDZ domain was requiredfor the distribution of RIM1 into the synaptic active zone.

FIG. 10 shows the involvement of CAST in neurotransmitter release andthe effect of CAST peptides on synaptic transmission. (a): The CASTpeptide sequences. RID, RIM1-interacting domain; scb RID, scrambled RID.(b): The effect of the peptides (5 mM each) on the binding of HA-RIM1 toimmobilized GST-CAST-4. Binding was inhibited by RID but not by RIDΔIWAor scb RID. (c) and (d): The effect of the CAST peptides (1 mM each inthe injection) on synaptic transmission. The presynaptic neuron wasstimulated every 20 seconds. The CAST peptides were introduced into thepresynaptic neuron at t=0. EPSPs from the representative experimentswith the injection are illustrated in (c). Normalized and averaged EPSPamplitudes are plotted from five experiments with RID (●), RIDΔIWA (Δ),or scb RID peptide (⋄) in (d).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is specifically described as follows.

Among the proteins of this invention, the protein comprising the aminoacid sequence described in SEQ ID NO: 2 has been identified as a proteinlocalized in synapses, as described in the Examples below. In the fieldof neuroscience, when, for example, the presence of a protein insynapses is to be proven, a protein whose presence in synapses hasalready been established as a synaptic marker is generally used.Furthermore, structural alterations of synapses are known to beassociated with diseases (Purpura, D. P., Dendritic spine “dysgenesis”and mental retardation, Science 186: 1126-1128, 1974; Geinisman Y., deToledo-Morrell L., Morrell F., Persina I. S., and Rossi M., Age-relatedloss of axospinous synapses formed by two afferent systems in the ratdentate gyrus as revealed by the unbiased stereological dissectortechnique., Hippocampus 2: 437-444 (1992). Therefore, it is thought thatthe proteins of this invention, and the DNA encoding these proteins,will be useful as synaptic markers in research and diagnostic fields.Furthermore, when the above-described structural alterations in synapsesare due to mutation and deletion of CAST, proteins of this invention andDNAs encoding these proteins can also be applied to the treatment fordisorders caused by structural alterations in synapses.

In addition, one aspect of proteins of this invention relates to thefunction of interaction with active zone proteins of synapses, such asRIM1 and Bassoon. Proteins of this invention in particular are capableof binding to RIM1 at the C terminus thereof, so as to localize RIM1 tosynapses. Therefore, proteins of this invention and DNAs encoding theseproteins may be used in the fields of research and medicine forlocalizing RIM1 to synapses.

It can be presumed that proteins have functionally identical mutants.Such mutants can be obtained by appropriate modification of the aminoacid sequences of the original proteins. Therefore, the proteins of thisinvention also include proteins that are localized to synapses and whichcomprise the SEQ ID NO: 2 amino acid sequence in which one or more aminoacids have been deleted, substituted, or added.

An amino acid substitution is preferably one in which the properties ofthe amino acid side-chain are conserved. A “conservative amino acidsubstitution” is a replacement of one amino acid residue belonging toone of the following groups having a chemically similar side chain withanother amino acid in the same group. Groups of amino acid residueshaving similar side chains have been defined in the art. These groupsinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

The number of amino acids that may be mutated is not particularlyrestricted, so long as the activity of the protein of this invention ismaintained. Generally, up to about 50 amino acids may be mutated,preferably up to about 30 amino acids, more preferably up to about 10amino acids, and even more preferably up to about 3 amino acids.Likewise, the site of mutation is not particularly restricted, so longas the mutation does not result in the disruption of the activity of theprotein of the present invention.

Protein amino acid sequences can be modified using known techniques,such as site-specific mutagenesis, to modify the DNA nucleotidesequences encoding the proteins, and then expressing the DNA containingthe modified nucleotide sequences. One skilled in the art can confirmthe localization of a protein to synapses by using fluorescent antibodymethods.

When the above-described mutant of the protein that is functionallyidentical to the protein as a synaptic marker, it is enough that themutant is able to maintain at least the property to be localized tosynapses as described above. On the other hand, when the mutant that isfunctionally identical to the protein is used for the interaction withRIM1, Bassoon and the like, it is necessary to select the mutant whichis capable of interacting with RIM1, Bassoon, etc. One skilled in theart can easily select such mutants capable of interacting with RIM1,Bassoon and the like, and, more specifically, the selection can beperformed with reference to Example 5.

Localization can also be confirmed using a fusion protein formed byfusing a protein of this invention with glutathione transferase (GST),His tag, or Green Fluorescent Protein (GFP).

As used herein, a “DNA” is an isolated nucleotide sequence the structureof which is not identical to that of any naturally occurring nucleicacid or to that of any fragment of a naturally occurring genomic nucleicacid spanning more than three genes. The term therefore covers, forexample, (a) a DNA which has the sequence of part of a naturallyoccurring genomic DNA molecule but is not flanked by both of the codingsequences that flank that part of the molecule in the genome of theorganism in which it naturally occurs; (b) a nucleic acid incorporatedinto a vector or into the genomic DNA of a prokaryote or eukaryote in amanner such that the resulting molecule is not identical to anynaturally occurring vector or genomic DNA; (c) a separate molecule suchas a cDNA, a genomic fragment, a fragment produced by polymerase chainreaction (PCR), or a restriction fragment; and (d) a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion protein. Specifically excluded from this definition are nucleicacids present in random, uncharacterized mixtures of different DNAmolecules, transfected cells, or cell clones, e.g., as these occur in aDNA library such as a cDNA or genomic DNA library.

The DNAs of the present invention encode the proteins of this inventionand include the DNA that comprises the nucleotide sequence set forth inSEQ ID NO: 1. This DNA has been sequenced according to the Exampledescribed below. Genes of the present invention are predicted to includegenes which encode identical products but which differ in theirnucleotide sequences, and genes that encode mutants with identicalfunctions. By modifying the nucleotide sequences it is also possible toobtain genes that encode mutants encoding identical products or haveidentical functions. Therefore, the DNAs of this invention also includeDNAs that have nucleotide sequences similar to that of SEQ ID NO: 1,that encode proteins localized to synapses, and that encode proteinscapable of binding to RIM or Bassoon. Such DNAs can be derived fromhumans, rats, mice, nematodes, and the like, however, are not limited tothese sources. Nematodes in particular are extremely useful as modelorganisms for studying the nervous system. In fact, the presence of aprotein of this invention in nematodes has already been proven (OhtsukaT. et al., J Cell Biol. 158(3):577-90 (2002).

In this invention, “DNA having a similar nucleotide sequence” includesDNAs that hybridize under stringent conditions to a DNA comprising anucleotide sequence complementary to that set forth in SEQ ID NO: 1, orDNAs that are homologous to the nucleotide sequences set forth in SEQ IDNO: 1 or NO: 3, such that the nucleotide sequence is 90% or more,preferably 95% or more, more preferably 98% or more, and even morepreferably 99% or more homologous.

Herein, stringent conditions refer to, for example, hybridization in4×SSC at 65° C. followed by washing in 0.×SSC at 65° C. for one hour.Alternative stringent conditions are hybridization in 50% formamidefollowed by washing in 4×SSC at 42° C. In addition, alternativeconditions are hybridization in PerfectHyb™ (TOYOBO) solution at 65° C.for two and a half hours followed by washing 1) in 2×SSC and 0.05% SDSat 25° C. for five minutes, 2) in 2×SSC and 0.05% SDS at 25° C. for 15minutes, and 3) in 0.×SSC and 0.1% SDS at 50° C. for 20 minutes (highlystringent conditions).

Homology as described herein is homology as calculated by the ClustalWmethod.

A portion of a DNA of this invention can be utilized as a primer orprobe to analyze a gene which encodes CAST, and the expression of thisgene. Furthermore, a portion of a DNA of this invention can be used as aprimer or probe in the detection of synapses. Thus, a DNA of thisinvention can be used in a reagent or kit for use in synapse-detectingresearch or diagnosis. In addition to a DNA of this invention, the kitof the present invention may contain a DNA encoding a peptide fragmentof CAST. Such CAST peptide fragments include fragments capable ofinhibiting the binding of CAST to RIM1. For example, a fragment at theC-terminus of CAST which comprises approximately ten amino acidsincluding the three amino acids (IWA) necessary for binding between CASTand RIM1 could be such a fragment. The fragment RID (set forth in SEQID: 6) as shown in the Examples below is preferable. However, suchfragments are not limited to this example. For example, the fragmentcapable of inhibiting the binding of CAST to RIM1 also includes afragment comprising the amino acid sequence set forth in SEQ ID NO: 6wherein one or several amino acids are deleted, substituted, or added,and having the activity to inhibit the binding of CAST with RIM 1.

In this invention, “a portion of DNA” refers to an oligonucleotide usedas a primer or probe and which comprises at least ten polynucleotidescorresponding to DNA sequences of this invention. Such polynucleotidescomprise preferably at least 15 nucleotides, and more preferably atleast about 20 to 30 nucleotides. Furthermore, polynucleotidescomprising larger macromolecules and full-length DNAs can also be usedas probes.

The DNA of this invention can be obtained using standard methods basedon identified nucleotide sequences. For example, the DNA can bechemically synthesized. Alternatively, it can be obtained by usingappropriately prepared primers and RT-PCR or mRNA prepared from neuronsand brain tissues.

Gene manipulation can be carried out according to the method describedin the reference: Maniatis et al, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, 1989.

There is no particular limitation as to the vectors of this invention solong as they are capable of stably maintaining the inserted DNA. Forexample, when E. coli is used as a host, pBluescript vector (Stratagene)or a similar vector is preferable as a cloning vector. When using avector in the production of a protein of this invention, expressionvectors are particularly useful. There are no particular limitations asto the specific type of expression vector, so long as it is capable ofexpressing the protein in vitro, in E. coli, in cultured cells, and inliving organisms. However, for example, the pBEST vector (Promega) ispreferable for in vitro expression; pET vector (Invitrogen) ispreferable for E. coli expression; pME18S-FL3 vector (GenBank AccessionNo. AB009864) is preferable for expression in cultured cells; and pME18Svector (Takebe Y. et al., Mol Cell Biol. 8:466-472, 1988) is preferablefor expression in living organisms. The DNA of this invention can beinserted into the vector using standard methods like the ligase reactionusing restriction enzyme sites (Current Protocols in Molecular Biologyedit. Ausubel et al. (1987). John Wiley & Sons, Section 11.4-11.11.

Transformants of the present invention can be obtained by transforminghosts with a DNA of this invention, thereby expressing a protein of thisinvention. A vector of the present invention can be introduced intovarious hosts without particular limitation, and a specific host can beselected in accordance with the objectives. Examples of hosts used forprotein expression include bacterial cells (e.g., Streptococcus,Staphylococcus, E. coli, Bacillus subtilis), fungus cells (e.g., yeast,Streptomyces, Aspergillus), insect cells (e.g., Drosophila S2,Spodoptera SF9), animal cells (e.g., CHO, COS, HeLa, C127, 3T3, BHK,HEK293, Bowes melanoma cells), and plant cells. Introduction of a vectorinto host cells can be performed using a known method such as calciumphosphate precipitation, electroporation (Current Protocols in MolecularBiology edit., Ausubel et al. (1987). John Wiley & Sons, Section9.1-9.9), lipofectamine (GIBCO-BRL) method, and microinjection.

The methods of production of the present invention are methods forproducing proteins of the present invention that are localized tosynapses. The methods include culturing transformants of the presentinvention, and collecting from the culture proteins which are localizedto synapses and expressed by the transformants.

The transformants may be cultured under conditions that enable theexpression of a protein of the present invention. The protein can berecovered from the culture by appropriately combining routine methodsfor protein purification, such as various types of chromatography,electrophoresis, and gel filtration. When the proteins of this inventionare expressed as fusion proteins with GST or His tag, they can bepurified using glutathione-Sepharose or nickel-Sepharose columns,respectively. Purified proteins can be used, for example, for antibodyproduction.

Antibodies can be produced by using the whole CAST of the presentinvention, or a part thereof, as an epitope. The resulting antibodiescan be used by one skilled in the art to detect synapses by knownmethods. For example, the method described in Example 4 could beemployed to detect synapses. Therefore, the antibody of the presentinvention can be used in a reagent or kit for use in synapse-detectingresearch or diagnosis. The kit of this invention may comprise a CASTpeptide fragment in addition to an antibody of this invention.

In this invention, the term “epitope” means a polypeptide's antigenicdeterminant, generally comprising at least six amino acids. It is wellknown in the art that polypeptides consisting of six amino acids canbind with antibodies (Published Japanese Translation of InternationalPublication No. Sho 60-500684). Antigenic peptides of the presentproteins mean polypeptides which comprise at least six, preferably atleast eight, more preferably at least about 15, and further morepreferably at least about 20 continuous amino acids based on the aminoacid sequences of the proteins of the present invention.

An Antibody of the present invention can be obtained from animals thathave been immunized with a protein of the present invention and preparedby a production method of the present invention. Polyclonal antibodiesare prepared from sera of the immunized animals described above.Monoclonal antibodies are prepared by fusing myeloma cells withantibody-producing cells obtained from the spleen or lymph nodes of theaforementioned immunized animals, and then selecting hybridomas thatproduce an antibody to the protein of the present invention with a highspecificity.

A protein of the present invention, which is obtained by a method ofproduction of the present invention, can be used as an immunogen.Alternatively, the immunogen may be a fragment or a peptide comprising apartial structure appropriately selected from the amino acid sequenceset forth in SEQ ID NO: 2. A variety of condensing agents, such asglutaraldehyde-,carbodiimide-, or maleiimide-activated esters can beused to prepare an antigen-carrier protein complex. Commonly-usedcarrier proteins such as bovine serum albumin, thyroglobulin, andhemocyanin may be used in the coupling reaction, generally in one- tofive-fold excess over the antigen.

The animals to be immunized include mice, rats, rabbits, guinea pigs,hamsters, etc. The animals are inoculated with the antigensubcutaneously, intramuscularly, or intraperitoneally. Prior toadministration, the antigen may be mixed with complete Freund's adjuvantor incomplete Freund's adjuvant, and administered usually once every twoto five weeks. Antibody-producing cells obtained from the spleens orlymph nodes of immunized animals are fused with myeloma cells andisolated as hybridomas. The myeloma cells used are derived from mice,rats, humans, etc. The myeloma cells are preferably obtained from thesame species as that providing the antibody-producing cells; however, itis also possible to use different species.

Cell fusion can be carried out, for example, according to the method ofKöhler and Milstein (Nature, 256, 495-497, 1975). Polyethylene glycol,Sendai virus, and so on can be used as fusion accelerators. However,cell fusion is usually carried out using polyethylene glycol (averagemolecular weight: 1,000 to 4,000) at a concentration of about 20% to50%, at 20° C. to 40° C., and preferably at 30° C. to 37° C. The ratioof antibody-producing cells to myeloma cells is generally from 1:1 to10:1.

Various immunochemical methods can be used in screening forantibody-producing hybridomas. For example, Enzyme-linked immunosorbentassays (ELISAs) in which microtiter plates are coated with a protein ofthis invention; enzyme immunoassays (EIAs) in which microtiter platesare coated with anti-immunoglobulin antibodies; immunoblotting using anitrocellulose transfer membrane following electrophoresis of samplescontaining a protein of this invention, etc.

Hybridoma clones are obtained by cloning samples in the wells of suchmicrotiter plates. This may be carried out by, for example, a limitingdilution assay. The hybridomas are screened and cultured, usually in amedium for animal cells (e.g., RPMI1640) that contains 10% to 20% fetalbovine serum supplemented with hypoxanthine, aminopterin, and thymidine(HAT). Clones thus obtained are introduced into the peritoneal cavity ofmice, preferably SCID mice, pre-administered with pristane. Ascitescontaining the monoclonal antibody in high concentrations are harvestedten to 14 days later and can be used as raw materials for antibodypurification. Alternatively, the clones are cultured, and the resultingculture can be used as material for antibody purification. Collection ofmonoclonal antibodies for immunoglobulin purification may be easilyperformed using known methods such as ammonium sulfate fractionation,PEG fractionation, ethanol fractionation, anion exchange, and affinitychromatography.

Qualitative and quantitative analyses of a protein of the presentinvention can be conducted in vivo by an immunological method using amonoclonal antibody obtained by the present invention. Knownimmunological methods (such as immunohistochemical staining, EIA,aggregation, competitive assays, or the sandwich method) can be appliedto biological samples which have been suitably treated as necessary, forexample, samples subjected to cell isolation or extraction.Immunohistochemical staining can be carried out, for example, bydirectly using labeled antibodies, or by indirect methods using labeledantibodies against that antibody. Any known labeling substances, such asfluorescent substances, radioactive substances, enzymes, metals, anddyes can be used.

A monoclonal antibody of this invention can be a Fab′ or Fab fractiondeprived of the Fc′ or Fc region, respectively, or it may be anaggregate of these fractions. The monoclonal antibody may also be achimeric antibody or a humanized antibody.

Pharmaceuticals that influence the expression of a protein of thepresent invention can be screened as described below:

A cell strain that expresses a protein of the present invention isselected by Northern blotting, RT-PCR, etc. Selection may also becarried out by using an antibody obtained by the above-described methodin fluorescent antibody assays, enzyme immunoassays, etc.

A selected cell strain is cultured in the presence of a testpharmaceutical. The effect of this test pharmaceutical on the expressionof a protein of the present invention is determined by quantifying thelevel of mRNA expression (using Northern blotting, slot blothybridization, RT-PCR, etc.), or by quantifying the level of proteinexpression (using fluorescent antibody assays, enzyme immunoassays,etc.).

Furthermore, a wide variety of pharmaceuticals may be screened moreeasily if the following method is used. Specifically, cDNA clonescapable of hybridizing to the 5′ region of cDNA for a protein of thepresent invention are selected from a human DNA library. These clonesare then inserted into an appropriate promoter screening system andclones with promoter activity are selected. Depending on the situation,the DNA region essential for the promoter activity may be narrowed atthis stage.

DNA comprising a promoter region of a protein of the present inventionis thus selected. A reporter gene is then constructed by inserting thisDNA upstream of DNA encoding enzymes whose activity can be easilyassayed, such as luciferase or alkaline phosphatase. The resultingreporter gene is transduced, together with appropriate resistance genessuch as Neo^(r) and Hyg^(r), into cells that can be cultured, such asHeLa cells. Cells are then selected using pharmaceuticals thatcorrespond to particular resistance genes. Thus, it is possible toestablish a cell strain that can be used to assay the activity of apromoter that expresses a protein of the present invention. The activityof the transduced enzyme in this cell strain is measured in the presenceof a pharmaceutical. Hence pharmaceuticals that affect the expression ofa protein of the present invention can be screened.

As an alternative screening system, compounds that affect theintracellular localization of a protein of the present invention may beselected. In one example of a screening method, an antibody obtained bythe above-described method is used to stain (using staining methods suchas fluorescence antibody assays, enzyme immunoassays, etc.) a cellstrain that expresses a protein of the present invention, or atransformant thereof, and through microscopic observation, measuring theinfluence of a test pharmaceutical on the intracellular localization ofa protein of the present invention. For example, this screening can beperformed according to the method of Examples 3 and 6.

Furthermore, in an alternative screening system, compounds that bind toa protein of the present invention can be selected. Compounds binding toa protein of the present invention are likely to affect the protein'sfunction. Equally, and as shown in the Examples, the protein of thepresent invention shows exocrine gland specificity in its distribution.Thus, if a compound is able to bind specifically to a protein of thepresent invention, it may express organ-specific actions. Furthermore,compounds binding to a protein of the present invention can be used insynapse detection. For example, synapses can be detected byadministering the labeled compound to, or bringing it into contact with,test subjects. Thus, the present invention also provides a method ofscreening for reagents for synapse-detecting research or diagnosis. Oneexample of a screening method uses a transformant of the presentinvention, a transformant's cytomatrix, an isolated protein of thepresent invention or a partial peptide thereof, etc. In this method, aprotein of the present invention is reacted with a test compound underappropriate conditions. The presence or absence of a bond between thetwo is then detected. Bonds can be detected by, for example, appropriateuse of labeled substances.

Furthermore, in another alternative screening system, compounds thatinhibit binding of a protein of the present invention to RIM1can beselected. As an example of such a compound, a peptide comprising theamino acid sequence set forth in SEQ ID NO: 6 is mentioned. Compoundsthat inhibit binding of a protein of the present invention to RIM1 canbe used for reducing a release of neurotransmitter such as acetylcholineand inhibiting synaptic transmission, as is shown in Example 6.

CAST structurally analyzed in the present invention is rat-derived.However, when human-derived CAST is used, the methods of the presentinvention entailing the analysis and screening of the CAST gene are alsoincluded in the scope of the present invention.

The present invention enables the detection and quantification of CASTand of mRNA encoding CAST. CAST is localized to synapses and tightlybound to the cytomatrix. Furthermore, it was revealed that 120 functionsas a protein scaffold for the localization of RIM1 to synapses,contributing on a molecular basis to the formation of the active zone.The present invention also provides markers, probes, and antibodies thatcan be used in synapse detection. Furthermore, the present inventionprovides methods of screening for compounds that can be used in synapsedetection.

All patents, published patent applications, and publications citedherein are incorporated by reference in their entirety.

Best Mode for Carrying Out the Invention

The present invention will be specifically described with reference toExamples below; however, it is not to be construed as being limitedthereto.

EXAMPLE 1

Cloning of DNA Encoding Cast

To identify proteins concentrated in the synaptic junction, crudemembrane (P2) and PSD fractions were prepared from rat cerebrum usingsucrose density-gradient centrifugation (Cohen et al., J. Cell Biol.74:181-203, 1977). Each fraction was dissolved in a urea-containingbuffer. These dissolved samples were then separately fractionated onMono Q anion exchange chromatography columns (Pharmacia). Followingelectrophoresis, each eluted P2 and PSD fraction was stained and bandpatterns were compared to identify 20 proteins concentrated in the PSDfraction. Some of these bands are shown in FIG. 1.

Mass spectrometry showed that most of these 20 proteins were identicalto proteins whose localization in synapses had been already reported.However, two of the proteins (the p500 and 120 proteins) did not havematches in mass spectrometry databases (see FIG. 1).

These proteins were then subjected to amino acid analysis, whichindicated that two p500-derived peptide sequences coincided with theinternal sequence of the CAZ protein, piccolo. Furthermore, four 120protein-derived peptide sequences were identical to the internalsequence of the KIAA0378 protein. Further analysis of the protein wasperformed since little is known about the function of the KIAA0378protein, in spite of the registration of its partial amino acidsequence. Herein, this 120 protein is referred to as “CAST”.

Based on the cDNA of the registered KIAA0378 (as described in Kikuno,R., Nagase, T., Waki, M. & Ohara, O. (2002) HUGE: a database for humanlarge proteins identified in the Kazusa cDNA sequencing project. NucleicAcids Res. 30, 166-168), primers set forth in SEQ ID NOs: 3 and 4 weredesigned and probes were prepared using PCR. Using this probe, a rathippocampus cDNA library was screened to obtain no less than ten clones.Of these, the two clones with the longest sequences were linked to formthe full-length cDNA. This nucleotide sequence is set forth in SEQ IDNO: 1. Its deduced amino acid sequence is set forth in SEQ ID NO: 2(illustrated in FIG. 2A).

The protein encoded by the full-length cDNA consists of 957 amino acidresidues with a few coiled-coil domains (FIG. 2A). The protein did nothave any acknowledged domain structures in particular. However it didhave a repetitive histidine sequence in its C-terminal region. In orderto confirm that the cloned cDNA actually encoded the full-length aminoacid sequence of the originally purified CAST, the cDNA was incorporatedinto an expression vector, and mRNA was then extracted to express theprotein in rabbit reticulocyte extracts (Promega). The antibody preparedin Example 2 also reacted with the endogenous CAST, and electrophoresisshowed that the protein encoded by the cDNA and the endogenous CAST hadalmost the same mobility (FIG. 2B). Therefore, it was concluded thatthis cDNA encodes the full-length amino acid sequence of CAST.

EXAMPLE 2

Preparation of an Antibody to CAST

A rabbit polyclonal antibody to CAST was prepared as follows: The geneencoding the fusion protein of glutathione S-transferase (GST) and aminoacids 180 to 380 of SEQ ID NO: 2 was incorporated into an expressionvector. The transformed vector was introduced into E. coli (JM109) andthe fusion protein thus expressed purified using a glutathione-Sepharosecolumn. Rabbits were then immunized with this purified protein.

EXAMPLE 3

Biochemical Examination of CAST Distribution

First, the CAST distribution in each organ was examined. Rats wereinfused with a PBS solution containing a protease inhibitor, and thentheir various organs were removed. Homogenates were prepared bydisintegrating each organ with a Teflon or polytron homogenizer. Afterthe quantification of proteins, 20 μg of each of the homogenates waselectrophoresed, transferred onto a nitrocellulose membrane, andsubjected to Western blotting using the above-described anti-CASTantibody. A band of approximately 120 kDa in MW was detected in thebrain homogenates (FIG. 3A). No band was detected in heart, spleen,lung, skeletal muscle, kidney, and testis, indicating that CAST isspecifically expressed in the brain.

Intracellular localization of 120 protein in the brain was thenexamined. After electrophoresis of membrane fractions separated bysubcellular fractionation (Cohen et al., J. Cell Biol. 74:181-203,1977), Western blotting was performed using the anti-CAST antibody. BothCAST and the NMDA receptor control were concentrated in the PSD fraction(FIG. 3B).

CAST was solubilized using a variety of surfactants. P2 fractions weretreated with CHAPS, NP40, Triton X-100, SDS, or DOC at room temperaturefor 30 minutes. Each sample was then ultracentrifuged (at 100,000×g) andseparated into supernatant and precipitate. Equivalent amounts ofsupernatant and precipitate were electrophoresed, and then subjected toWestern blotting using the anti-CAST antibody.

CAST was not solubilized in non-ionic surfactants such as CHAPS, NP40,and Triton X-100, and was recovered in the precipitate (FIG. 3C).However, the 120 protein was solubilized by ionic surfactants, SDS andDOC, and thus recovered in the supernatant. These results indicate thatCAST is localized to the synaptic junction and tightly bound to thecytomatrix.

EXAMPLE 4

Tissue Distribution of CAST

Since the above-described biochemical data suggested that CAST is asynaptic protein, 120 protein localization to the synapses in actualtissues was examined.

Mouse hippocampal regions were immunohistostained using anti-CASTantibody, and the presynaptic synaptic vesicle protein, synaptotagmin I,was used as a control. The strongest CAST signal was observed in the CA3region of the hippocampus (FIG. 4A). This region is referred to as thestratum lucidum and is where synapses are formed between the mossy fibernerve terminals and the dendrites of pyramidal cells, thus indicatingthe localization of CAST to synapses.

Using a primary culture of neurons from the rat hippocampus, thelocalization of CAST to synapses was further investigated. On embryonicday 19, hippocampi were excised from rat cerebra and cells were treatedwith trypsin before being cultured in B-27 medium (Gibco). Cells on the21^(st) day of culture were fixed in 4% paraformaldehyde, treated with0.2% Triton X-100, co-incubated with the anti-CAST antibody and eitherthe anti-Bassoon antibody or the anti-PS-95 antibody, and then thelocalization of each protein was detected using a secondary antibody.Images of the synaptic pattern of CAST showed it was expressed togetherwith Bassoon and PS-95 along the dendrite (FIG. 4B), strongly indicatingthe localization of CAST to synapses.

Furthermore, analysis using immunoelectron microscopy revealed that CASTis localized very closely to the presynaptic active zone of thehippocampal CA3 region (FIG. 4C).

EXAMPLE 5

Identification of the Binding Protein of CAST

Proteins binding to CAST were identified in order to analyze thefunction of CAST in the active zone.

Immunoprecipitation of the P2 fraction with the anti-CAST antibody sawCAST co-precipitate with active zone proteins, RIM1and Bassoon (FIG. 5).Bassoon's molecular weight of 4,000,000 or more renders its biochemicalanalysis problematic, and thus the binding of 120 with RIM1 was furtheranalyzed.

In 1997, RIM1 was isolated as a target protein for small G proteinRab3A; however, its function is still unknown (Wang et al., Nature. 388:593-598, 1997). Recently reported RIM1 knockout mice revealed that RIM1plays an extremely important role in synaptic plasticity. However, thesereports did not show the initially expected involvement of RIM1 inactive zone formation (Castillo P. E. et al., Nature 415:327-330, 2002;Schoch S. et al, Nature 415:321-326, 2002).

When GST fusion proteins of RIM1 and P120 proteins were prepared todetermine the mutual binding domains, it was revealed that the PDZdomain of RIM1 and the C-terminus of CAST directly bind to each other(FIG. 6). In particular, the terminal Ile-Trp-Ala (IWA) amino acids wererevealed to be essential to this binding. The above-described resultsindicated that CAST functions as a protein scaffold for RIM1 in theactive zone.

A primary culture of rat hippocampal neurons was then used to examinewhether CAST actually functions as a protein scaffold for RIM1 inneurons. When CAST and RIM1 were co-expressed in neurons, they wereco-localized with the synaptic marker protein, synaptophysin (FIGS. 7and 8). When CAST was expressed alone, the same results were obtained.Furthermore, a mutant CAST, in which the C-terminal IWA amino acidsnecessary for binding RIM1 had been deleted, could also be localized tosynapses. Thus it was revealed that RIM1 is not required for thelocalization of CAST to synapses.

Next, whether CAST is necessary for the localization of RIM1 to synapseswas examined. When expressed alone, RIM1 was co-localized with Bassoonto synapses (FIG. 9). However, mutant RIM1, deprived of the PDZ domainnecessary for binding to CAST, was not localized to synapses. However,when RIM1 was co-expressed with CAST, both the PDZ domain and CAST wereco-localized with Bassoon to synapses. The above-described resultsproved that RIM1 localizes to the synapses by binding, via its PDZdomain, to the IWA sequence of CAST.

The present inventors found that CAST is a very important active zoneprotein in determining the localization of RIM1. The formation of amolecular complex based on the interaction between active zone proteinsis likely to contribute a great deal to the molecular basis of activezone formation.

EXAMPLE 6

Involvement of RIM1 Binding to CAST in Neurotransmitter Release

In the next set of experiments, the present inventors examined whetherthe binding of RIM1 and Bassoon to CAST is involved in neurotransmitter(acetylcholine) release. The present inventors used superior cervicalganglion neurons (SCGNs) in culture for this purpose, because i)peptides or proteins can be readily introduced into the relatively largepresynaptic cell bodies using microinjection, ii) the injected materialscan rapidly diffuse to nerve terminals forming synapses with adjacentneurons, and iii) the effects of the stimulated release of acetylcholinecan be accurately monitored by recording the excitatory postsynapticpotentials (EPSPs) evoked by action potentials in the presynapticneurons (Mochida S. et al., Proc Natl Acad Sci U.S.A. 95(24):14523-8,1998).

The present inventors first prepared the RIM-interacting domain (RID)peptide, and the RIDΔIWA and scrambled RID peptides as controls (FIG. 10a). The last three amino acids (IWA) are critical for the binding ofRIM1 to CAST (Ohtsuka T. et al., J Cell Biol. 158(3):577-90, 2002). Invitro binding assays revealed that RID, but not RIDΔIWA or scrambledRID, inhibited the binding of RIM 1 to CAST (FIG. 10 b).

The present inventors next microinjected these peptides into presynapticSCGNs. RID inhibited synaptic transmission (FIG. 10 c and FIG. 10 d). At70 minutes after RID injection, EPSP amplitude was reduced by −33+9.1%(n=5). In contrast, neither RIDΔIWA nor scrambled RID produced asignificant decrease in EPSP amplitude (p=0.025, 0.023 unpaired t-testat 70 minutes after the injection of RID versus RIDΔIWA, and RID versusscrambled RID, respectively).

These results indicate that CAST dynamically binds RIM1and that thisdynamic binding is necessary for neurotransmitter release. It has beenshown that the direct binding of RIM1 and Munc13-1 is involved in thepriming of synaptic vesicles (Betz A. et al., Neuron. 30(1): 183-96,2001) and that the localization of RIM1 at the CAZ appears to beCAST-dependent (Ohtsuka T. et al., J Cell Biol. 158(3):577-90, 2002).The inhibition of RIM1 binding to CAST may affect the RIM1-Munc13-1pathway, presumably by mislocalization of RIM 1 at the active zone,resulting in reduction of neurotransmitter-release.

1. An isolated protein selected from the group consisting of: (a) aprotein having the amino acid sequence set forth in SEQ ID NO: 2; and(b) a protein which binds to Rab3A-interacting molecule (RIM1), saidprotein (i) has an amino acid sequence with one to nine amino aciddeletions, substitutions, or additions in the amino acid sequence of SEQID NO: 2, and (ii) contains Ile-Trp-Ala at the C-terminus.
 2. Aninsulated protein encoded by DNA selected from the group consisting of:(a) an isolated DNA that hybridizes under stringent conditions to a DNAthat has a nucleotide sequence complementary to the nucleotide sequenceset forth in SEQ ID NO: 1, wherein the stringent conditions arehybridization at 65° C. for two and a half hours followed by washing in2×SSC and 0.05% SDS at 25° C. for five minutes; and (b) an isolated DNAhaving a nucleotide sequence with 95% or more homology to the nucleotidesequence set forth in SEQ ID NO: 1 and encoding a protein which binds toRIM1, and wherein said protein contains Ile-Trp-Ala at the C-terminus.3. An isolated DNA selected from the group consisting of: (a) a DNAencoding a protein having the amino acid sequence set forth in SEQ IDNO: 2, and (b) a DNA encoding a protein which binds to RIM1, saidprotein (i) has an amino acid sequence with one to nine amino aciddeletions, substitutions, or additions in the amino acid sequence of SEQID NO: 2, and (ii) contains Ile-Trp-Ala at the C-terminus.
 4. Anisolated DNA encoding a protein having the amino acid sequence set forthin SEQ ID NO: 2 and having nucleotides 59 to 2929 of the nucleotidesequence set forth in SEQ ID NO:
 1. 5. An isolated DNA that hybridizesunder stringent conditions to a DNA that has a nucleotide sequencecomplementary to the nucleotide sequence set forth in SEQ ID NO: 1,wherein the stringent conditions are hybridization at 65° C. for two anda half hours followed by washing in 2×SSC and 0.05% SDS at 25° C. forfive minutes.
 6. An isolated DNA having a nucleotide sequence with 95%or more homology to the nucleotide sequence set forth in SEQ ID NO: 1and encoding a protein which binds to RIM1, and wherein said proteincontains Ile-Trp-Ala at the C-terminus.
 7. An isolated DNA encoding aprotein having the amino acid sequence set forth in SEQ ID NO: 2 andhaving the nucleotide sequence set forth in SEQ ID NO:
 1. 8. Arecombinant vector comprising the DNA of any one of claims 3, 4, 5, 6and
 7. 9. An isolated host cell transformed with the DNA of any one ofclaims 3, 4, 5, 6 and
 7. 10. A method for producing a protein encoded byan isolated DNA of any one of claims 3, 4, 5, 6 and 7 localized tosynapses, wherein said method comprises the steps of: (1) culturing ahost cell comprising said DNA; and (2) collecting the protein from theculture.